Project Summary The long-term goal of my research is to comprehensively characterize the RNA structurome, here defined as the collection of all RNA structures and RNA-RNA interactions in living cells. RNA structures represent an important, yet under-appreciated, layer of genetic information that is essential for the interpretation and execution of the genomic blueprint. In this application for career development award, I have outlined research strategies that range from development of methods for RNA structure determination to functional characterization of RNA structures, which will eventually lead to applications to human diseases, the ultimate goal of genome medicine. RNA structures and interactions are fundamental to RNA's diverse functions, such as guiding, scaffolding and catalysis. Not surprisingly, genetic alternations of RNA structures or helicases (enzymes that remodel RNA structures) underlie many human diseases, including various cancers. RNA viruses cause some of the most deadly human infections, and viral genomic RNA structures control critical steps in their lifecycle. However, only a few RNA structures have been determined due to lack of proper methods. To address this issue, I developed PARIS, a psoralen-crosslinking based method for high throughput mapping of RNA duplexes in living cells at single-molecule level with near base-pair resolution (Lu et al. 2016 Cell). In this proposal, I will further increase the capabilities of PARIS by developing new high-efficiency photochemical crosslinkers (high solubility psoralens and bifunctional carbazoles) and resolution-refining enzymatic strategies. Based on the PARIS-determined structures, and integrating clustered interaction sites of RNA-binding proteins, I will test the hypothesis that RNA domains are structural and functional units of long non-coding RNAs. PARIS-determined structures also made it possible to conduct global screens for their functions using synthetic translation reporters. Systematic mutagenesis and screening of protein effectors will be used to dissect the mechanism of function for regulatory structural elements. My previous training in a wide variety of topics that span computational and experimental biology set a solid foundation for my future success. To achieve my career goals goals, I have designed further training plans that will equip me with the critical skills in chemical biology, computational analysis and high throughput function screening. On one hand, I will obtain training through collaborations with Stanford faculty members, including my mentor Dr. Howard Chang an expert in chromatin and RNA biology, Dr. Eric Kool, an expert in photochemistry, Dr. Rhiju Das, an expert in computational biochemistry and RNA structure analysis, Dr. Peter Sarnow, an expert in translational control and RNA virus. The work conducted at Stanford will be well supported by the superb academic environment that includes all the intellectual interactions and necessary facilities. On the other hand, my team of collaborators includes international experts such as Dr. Edith Heard from the Curie Institute in France, who specializes in stem cell biology and X chromosome inactivation, and Dr. Eran Segal at the Weizmann Institute in Israel, who specializes in genomics and high throughput screens. Together, my support network provides me with the essential assistance for both scientific growth and career development. In summary, the proposed studies will deliver a set of powerful tools to the broad RNA community for RNA structure determination, elaborate and validate the new concept of high-level RNA modularity, and provide new insights into RNA functions through multi-scale interrogation of RNA structures, from domains to single base pairs. Comprehensive characterization of the RNA structurome will uncover the regulatory mechanisms that translate the genome to phenotype, in both physiological and disease contexts. This training plan will not only establish a successful research program but will also facilitate my transition to an independent faculty position in a top research institute.